The Efficacy of Therapeutic DNA Vaccines Expressing the Human Papillomavirus E6 and E7 Oncoproteins for Treatment of Cervical Cancer: Systematic Review

Cervical cancer is recognized as a serious public health problem since it remains one of the most common cancers with a high mortality rate among women despite existing preventative, screening, and treatment approaches. Since Human Papillomavirus (HPV) was recognized as the causative agent, the preventative HPV vaccines have made great progress over the last few years. However, people already infected with the virus require an effective treatment that would ensure long-term survival and a cure. Currently, clinical trials investigating HPV therapeutic vaccines show a promising vaccine-induced T-cell mediated immune response, resulting in cervical lesion regression and viral eradication. Among existing vaccine types (live vector, protein-based, nucleic acid-based, etc.), deoxyribonucleic acid (DNA) therapeutic vaccines are the focus of the study, since they are safe, cost-efficient, thermostable, easily produced in high purity and distributed. The aim of this study is to assess and compare existing DNA therapeutic vaccines in phase I and II trials, expressing HPV E6 and E7 oncoproteins for the prospective treatment of cervical cancer based on clinical efficacy, immunogenicity, viral clearance, and side effects. Five different DNA therapeutic vaccines (GX-188E, VGX-3100, pNGVL4a-CRT/E7(detox), pNGVL4a-Sig/E7(detox)/HSP70, MEDI0457) were well-tolerated and clinically effective. Clinical implementation of DNA therapeutic vaccines into treatment regimen as a sole approach or in combination with conservative treatment holds great potential for effective cancer treatment.


Introduction
Cervical cancer is a largely preventable cancer of the cervix, which is the narrow part of the lower uterus that connects to the vagina. According to the World Health Organization (WHO) statistics, cervical cancer became the fourth most frequent cancer in women in 2018, with 570,000 cases, which represent 6.6% of all female cancers worldwide [1]. Similarly, cervical cancer is the 2nd most common type of cancer among females, and the 4th most common cause of cancer-related deaths (8.5%) among women in Kazakhstan [1].
Previous studies have established the strong causative association between persistent infection with certain high-risk Human Papillomavirus (HPV) types and the development of cervical cancer [2]. HPV is a small, non-enveloped deoxyribonucleic acid (DNA) tumor virus, which primarily affects human vaginal and oral mucosa [2]. There are more than Therefore, the aim of this article is to assess and compare existing DNA therapeutic vaccines, which are evaluated in phase I and II trials, expressing HPV E6 and E7 oncoproteins for the prospective treatment of cervical cancer based on clinical efficacy, immunogenicity, viral clearance, and side effects.

Materials and Methods
This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews (PRISMA) statement [11]. The study was registered in the PROSPERO database and confirmed with a registration code of CRD42021251476.

Systematic Literature Search and Eligibility Criteria
Articles were manually searched using databases as PubMed/MEDLINE, Google Scholar, and clinicaltrials.gov published in English from the year 2010.
The search was narrowed by using "Cervical cancer OR Cervical Intraepithelial Lesion AND DNA therapeutic vaccines", "DNA therapeutic vaccines AND E6 OR/AND E7 oncoproteins". The selected studies were independently reviewed for inclusion eligibility by two reviewers (Akhatova and Aimagambetova) using standardized data collection forms. The following data were collected from the studies: the author, year of publication, number of study participants, vaccine administration strategies, and the main outcomes (clinical efficacy, viral clearance, immunogenicity, adverse events). Any discrepancy in the assessment of articles was resolved by discussion and consensus, as well as input from the third and fourth reviewers (Chan and Azizan).
The articles were selected to meet the following eligibility requirements to be included in the study: (1) research article, (2) human subject research, and (3) the study of DNA therapeutic vaccines targeting HPV E6 and E7 oncoproteins. The presence of the following did not allow for the study to be included: (1) reviews and case reports, (2) irrelevance to cervical cancer or CIN, (3) mouse model studies, (4) articles on preventative HPV vaccines, and (5) the use of the inappropriate methodology. Abstracts lacking full information about predefined criteria were excluded without further review.
The types of studies included were phase I and phase II clinical trials that were initiated and completed between 2003 and 2017, studying the clinical efficacy of DNA therapeutic vaccines expressing HPV E6 and E7 oncoproteins for the treatment of cervical intraepithelial lesions of grades 2 and 3 both newly diagnosed and recurrent malignancies. The treatment of the lesion may or may not be followed by conization or loop electrosurgical excision procedure. The study population was female patients aged 18 or older with histopathologically diagnosed CIN of grades 2 and 3, known to be caused by HPV 16 and/or HPV 18 based on polymerase chain reaction (PCR) amplification results. The main outcomes of the review were clinical efficacy based on the lesion regression, viral load reduction, immunogenicity, in particular, HPV E6 and E7 specific CD8+ T cell response, and AEs after vaccination.

Study Identification and Selection
During this study, 120 articles were identified through PubMed/MEDLINE and Google Scholar searching platforms ( Figure 1). Eighty-three articles were excluded based on the abstract, representing literature reviews. From the remaining 37 articles, 24 articles were excluded at this stage: 11 studies were mouse model-based, 9 studies were not addressing the study question, 2 studies were regarding the preventative vaccines, and 2 studies included additional oncogenes in the development of therapeutic vaccines. We included 6 studies performed between 2003 and 2017 in our systematic review after the exclusion of 7 articles studying the effects of therapeutic vaccines with either viral, bacterial, or peptide vectors [12][13][14][15][16][17]. These six studies represented the work completed in the United States of America, Korea, Estonia, South Africa, India, Canada, Australia, and Georgia [12][13][14][15][16][17] (Table 1). Completed NCT02139267

GX-188E [13]
Subjects-20-50 year old women with HP diagnosed HPV16/18-associated CIN3 were vaccinated in a series of three injections IM using EP device in deltoid muscle at weeks 0, 4 and 12. A standard 3+3 dose escalation scheme was followed and dose levels of 1, 2 and 4 mg (2 + 2 mg) were tested within 36 weeks of follow up.
Therapeutic vaccines were evaluated based on clinical efficacy (histopathological regression of the lesion to CIN < 1), viral clearance, immunogenicity, and adverse events after the vaccination. Subjects, female patients aged 18 or older with histopathologically diagnosed cervical intraepithelial neoplasia of grades 2 and 3, known to be caused by HPV 16 and/or HPV 18, received DNA therapeutic vaccines in different dose formulations and were evaluated generally within 20 weeks (36 weeks in extended trial groups) for the effects of vaccines mentioned above. Study populations ranged from 10 patients to 167, median age mostly being 21-30, except for Hasan et al. (2020) study, where the median age of participants was 51.50 years old due to more advanced stages of cancer [17].

Clinical Efficacy
Clinical efficacy was evaluated according to the histopathological regression to CIN ≤1, which is less than one-third of the thickness of the cervical epithelium, on a colposcopyguided biopsy 15, 20, or 36 weeks after the first injection. All six studies [12][13][14][15][16][17] report tumor size decrease to some extent ( Table 2).
GX-188 E in phase I trial by Kim et al. [13] showed a 78% success rate of complete response both histologically and virologically. The same vaccine in the phase II trial by Choi et al. (2019) resulted in histopathological regression to CIN < 1 in 52% of patients 20 weeks and 67% of patients 36 weeks after the first dose [12]. MEDI0457 by Hasan et al. showed 87.5% of complete response to the vaccine and 1 patient had a partial response to the treatment [17]. pNGVL4a-CRT/E7(detox) [15] and pNGVL4a-Sig/E7(detox)/HSP70 [16] vaccines both showed a similar response rate of around 30%.

Viral Load Clearance
Viral load was measured by means of PCR amplification to assess the clearance of HPV DNA from the cervical biopsy after vaccination. Choi et al. established that HPV clearance was associated with the histopathologic regression as 77% of regressors had no trace of HPV DNA, while only 12% of non-regressors had no viral load in the tissue biopsy [12]. Kim et al. results show that GX-188E takes time to clear off the virus [13]. MEDI0457 [17] and VGX-3100 [14] report the association between viral clearance and tumor size reduction, whilst pNGVL4a-CRT/E7(detox) [15] did not result in any difference between pre-and post-treatment viral load.

Immunogenicity
Immunogenicity is one of the key features of the therapeutic vaccines as it represents the potential of the vaccine to induce virus-specific T cell response, in particular HPV E6 and E7 specific CD8+ T cell immune response. IFN-γ response was measured by means of ex vivo ELISpot assay with cryopreserved and thawed peripheral blood mononuclear cells (PBMCs) at pre-and post-treatment stages. The vaccine response is considered positive when the increase in T-cell frequency was at least three times greater compared to the study entry measurement. GX-188E both in phase I [13] and phase II [12] studies showed a significant increase in IFN-γ response, which was correlated with the histopathologic regression and viral clearance. Moreover, an E6 specific response was more pronounced than E7 specific [12,13]. VGX-3100 induced 9.5 times greater IFN-γ response in the treatment group compared to the placebo, which lasted as long as 24 weeks post-vaccination [14]. On the contrary, MEDI0457 induced a greater response to E7, particularly in newly diagnosed cohort 1 that persisted up to 48 weeks [17]. In cohort 1, 4 of 7 patients exhibited IFNγ-producing spots exceeding 100 SFU/10 6 PBMC, whereas no patients produced similar responses in cohort 2 [17]. pNGVL4a-CRT/E7(detox) and pNGVL4a Sig/E7(detox)/HSP70 showed minimal dose-dependent immune response, which was remarkable from the unvaccinated group [15,16]. GX-188E-well-tolerated. The AEs relating to the injection site-pain, erythema, induration, and swelling/edema in both groups; pain was the most common AE (occurring in 94.4% and 100.0% in the 1 and 4 mg GX-188E groups, respectively) One patient was lost to follow up due to pregnancy (1 mg GX-188E group). Lack of control/placebo group. The selection bias-patients recruited into the study were diagnosed with CIN 3 only. The attrition bias-20/72 participants withdrew from the study due to various reasons. The confirmation bias-in the discussion part, authors concluded that immunologic response and HP regression had weak association. However, earlier in the results they mentioned an association between HP regression and systemic immune response.
All subjects exhibited a marked increase in the vaccine-induced E6-and E7-specific IFN-g ELISPOT response compared with the background level before vaccination. Vaccine-induced cellular immune responses became progressively stronger in all patients during GX-188E vaccination. The response against the E6 antigen was more vigorous than against E7 as determined by the magnitude of response (69-89% against E6 versus 11-31% against E7 at VF1). GX-188E vaccination-induced E6/E7specific memory T-cell response can be maintained for at least 24 weeks post last vaccination. Apart from patient A04, GX-188E vaccine elicited activation of both HPV16-specific CD4 and CD8 T cells. The amount of Th1 effector cytokines, such as IFN-γ, IL-2 and tumour necrosis factor α (TNF-α) increased after vaccination in most of the patients (median 49.9−, 13− and 22.9−fold increases for IFN-γ, IL-2, and TNF-α, respectively). In post-hoc immunological analyses, T-cell responses to HPV-16 and HPV-18 E6 and E7 peaked at week 14 for VGX-3100 recipients, with a 9.5 times greater median response than in placebo (p < 0·0001). VGX-3100 elicited significantly increased frequencies of antigen-specific, activated CD8+ T cells, identified by cell surface expression of CD137, that also expressed perforin compared with placebo (p = 0·001). VGX-3100 recipients with HP regression and viral clearance developed antibody responses to both HPV-16 and HPV-18 E7 that were significantly higher than for non-regressors, at the time of peak response (post-dose 3) but also as early as post-dose 2 and as late as week 24.

None
Skewing of the population towards more severe disease and older age. 92.8% of the participants had genotype of HPV 16 + at the entry. The attritition bias-18 patients in treatment group and 6 patients in control group were excluded from the study due to different reasons. HPV genotyping, which was based on the cervical swabs, included the possibility of only HPV16 or/and HPV18. Therefore, mixed infection study group could be underestimated. The confirmation bias-Vaccination induced HP regression and viral clearance in about 40% of women with CIN2/3 positive for HPV-16 or HPV-18, whereas surgical excision would have eliminated the dysplastic tissue in 85-90% of women. No differences between pre-and post-vaccination viral loads in any of the treatment cohorts.
Immune response to E7 was minimal and was not significantly different than response to E6. Intraepithelial CD8+ T cell infiltrates increased after vaccination in intralesional administration cohort (p = 0.0313).

None
This was a small phase I trial designed to primarily evaluate the feasibility and safety of pNGVL4a-CRT/E7(detox). Only HPV 16 positive CIN patients were included in the study. The majority of these patients were Caucasians. Patients were required to have a hemoglobin of 9 g/dL or greater. The selection bias-anemia is considered strong prognostic factor. The ND10 PMED has a reduced number of components to ease large-scale manufacturability, compared to previously used ND5.5. This could potentially lead to discrepancies in results due to device error.
NA E7 specific T cell response was identified in 3/15 patients: • 1 patient-response increased subsequent to vaccination at week 15 E6 specific T cell response: 5/15 patients. Overall, responses to E6 were not of greater absolute magnitude in regressors compared with non-regressors at either of the two time points. (p = 0.4228 and p = 0.4964, respectively). At 6 months response to E7 was detected in 5/9 patients (55.6%) in highest dose cohort.

None
This was a small phase I study-15 patients only. No masking. Follow up period was 19 weeks, whereas the average follow-up period in selected studies was 36 weeks. Vaccine targets specifically HPV16 E7 oncoprotein, without HPV 18,or E6 oncoprotein. Local and systemic AEs were assessed by patients, which may result in self-reporting bias such as social desirability or recall bias.

MEDI0457 [17]
All cohort 1 patients remain alive with no evidence of disease clinically or by PET/CT. Of the cohort 2 patients: 1 remains free of disease. The estimated PFS at 12 months was 88.9% overall, 100% in cohort 1, and 50% in cohort 2. 7/8 patients achieved a complete response (6/7 in cohort 1 and 1/3 in cohort 2), and 1 (cohort 1) achieved partial response (decreased or stable hypermetabolic activity after CRT+MEDI0457) after completion of the immunization series.
All patients cleared detectable HPV DNA at week 16 after immunizations. 5/6 patients cleared HPV RNA by in situ hybridization at the completion of immunization.
Expression of PD-L1 on panCK+ tumor cells, CD68+ macrophages, and CD8+ T cells in serial biopsy specimens: Too small study (n = 10) population, which does not allow for generalization of the results and drawing conclusions. Study included several histologic diagnoses-squamous cell carcinoma, adenocarcinoma, adenosquamous cell carcinoma of the cervix with various prognosis. The confirmation bias-patients received a vaccine 2 to 4 weeks after chemoradiation, which could impact the vaccine effect on organism. It is unclear whether longer period of recovery would result in better outcome. Dosing and timing regimen of MEDI0457 was based on studies of preinvasive cancer, thus the applicability of the regimen for invasive cancer types is questionable [15]. There was no control group of "chemoradiation only" in order to assess the sole effect of vaccination.

Toxicity/Adverse Events
Overall, all vaccines were well-tolerated without vaccine-related serious adverse events. The most common adverse events were injection site pain and erythema, as well as constitutional symptoms (malaise, myalgia, and headache) [12][13][14][15][16][17]. No serious adverse events (Grade 3/4) related to the vaccination were reported. No dose-limiting toxicities were observed.

Discussion
This systematic review summarizes the findings of phase I and phase II clinical trials investigating the treatment of patients with histopathologically diagnosed CIN associated with HPV 16 or/and HPV 18 with DNA therapeutic vaccines. Six studies have demonstrated immunologic response in the form of lesion size regression, viral clearance, and increased T cell response of five different DNA vaccines-GX-188E (phase I and phase II), VGX-3100, pNGVL4a-CRT/E7 (detox), pNGVL4a-Sig/E7 (detox)/HSP70, MEDI0457. Vaccines were plasmid DNA encoding for either non-oncogenic E6/E7 or both, and chaperonin proteins such as HSP 70 and Calreticulin for the enhancement of the uptake by antigen-presenting cells, and MHC class I processing and presentation. MEDI0457 [17] had the same plasmid formulation as VGX-3100 [14] combined with plasmid encoding IL-12. All vaccines were well tolerated by patients, leading to only grade 1 or less systemic and local side effects.
Previous reviews have studied various existing therapeutic vaccines including live vectors, plant-based, protein, whole cell, and combinatorial vaccines [18]. This is the first systematic review of DNA therapeutic vaccines against cervical cancer expressing HPV16 and HPV18 E6 and E7 oncogenes. The feasibility of production, storage, and transportation, cost-effectiveness, the capability of multiple immunizations, and targeting different costimulatory genes provided the rationale for the study of DNA therapeutic vaccines [18]. However, comparatively weak immunogenicity and the risk of integration into the host genome are the main concerns, which could be addressed by modification of E6 and E7 to abolish its transformative capacity [18]. There are approaches of boosting the potency of DNA vaccines, such as increasing the number of antigen-expressing dendritic cells (DCs) by using a gene gun delivery method, enhancing antigen processing and presentation in dendritic cells via codon optimization, and improving the DCs and T-cell interaction [18]. These strategies were used in our selected studies, which led to increased antigen-specific, activated CD8+ T cell response in all of them. Patients with CIN2/3 were more likely to induce E6 and E7 specific CD8+ immune response, according to the IFNgamma ELISPOT results, compared to the invasive cervical cancer [17]. According to Hasan et al., diminished immune response in more advanced disease stages is associated with immune exhaustion, the effect of chemoradiation and selection of patients with diminished immunity against HPV [17]. The strongest evidence of the immunogenicity of DNA therapeutic vaccine VGX-3100 was observed by the increased intensity of CD8+ infiltrates in histopathologically regressed patients compared to the placebo group with regressed lesions [14].
DNA therapeutic vaccines were also assessed based on their clinical efficacy, i.e., the ability to induce cervical lesion regression. The regression to ≤CIN1 among study participants was observed in all studies with significantly varying degrees. The study of VGX-3100 vaccine with both treatment and placebo groups showed a response rate of 49.5% vs. 30.6%, respectively [14]. Meanwhile, GX-188E vaccine has resulted in histopathological regression in 67% of patients in both phase I study and phase II studies [12,13]. Choi et al. [12] have observed an enhanced response to GX-188E over time up to 83% among those with cervical lesions <50%, probably due to the enhanced memory T cell-driven therapeutic effect. The difference in clinical benefit between VGX-3100 and GX-188E could be explained with the recruitment of CIN3 HPV-positive patients only, the lack of placebo group, and the small number of participants in the latter. pNGVL4a-CRT/E7 (detox) and pNGVL4a-Sig/E7 (detox)/HSP70 had the lowest clinical efficacy of approximately 30% response rate among all [15,16]. However, the effect of these two vaccines on the lesion regression is questionable, as this rate is similar to spontaneous remission rate over a 15-week period [15].
It was established that women, after excision of the cervical lesion, are more likely to have a relapse; therefore, viral clearance is a key factor of vaccine efficacy [18]. VGX-3100, GX-188E, and MEDI0457 effectively cleared detectable HPV DNA, which was significantly associated with histopathological regression [12][13][14]17]. In contrast, pNGVL4a-CRT/E7 (detox) has not resulted in viral load reduction [15].
Nevertheless, there are several limitations to this study. Firstly, limited data exist on the topic of DNA therapeutic vaccines, as not a single therapeutic vaccine against cervical cancer was approved. All these clinical trials were in either the phase I or phase II stage of assessing the efficacy and safety in humans. Secondly, the majority of studies enrolled a small number of participants without masking, stratification, or the control group, which poses a potential risk for bias. As vaccines investigated in this study had different structural designs, it was not feasible to make a statistical analysis of vaccine outcomes; therefore, qualitative analysis was performed overall.

Conclusions
As it was stated by WHO, a global strategy to accelerate the elimination of cervical cancer, 90-70-90 targets for prevention, screening, and treatment are the key to success. Preventative bivalent and quadrivalent HPV vaccines have undergone significant advancement in development and implementation. However, these preventative vaccines do not elicit a therapeutic effect but could be used as an adjuvant to surgical treatment. DNA therapeutic vaccines represent a potentially safe and novel approach to cervical cancer treatment. The main goal of this review was to discuss the effectiveness of existing DNA therapeutic vaccines against cervical cancer expressing HPV 16/18 oncoproteins E6 and E7. The idea of DNA therapeutic vaccines is inducing an adaptive immune response and immunologic memory via the expression of tumor antigens and activation of antigen-presenting cells.
DNA therapeutic vaccines are currently undergoing clinical trials to improve the potency of therapeutic vaccines and clinical efficacy using strategies as a modifying route of administration, adjuvant therapy, prime-boost regimen, and co-administering with other drugs for a synergistic effect. Nowadays, despite the treatment of locally advanced disease with chemoradiation, patients have a high recurrence rate and a poor 5-year survival rate, estimated at 50% and 70%, respectively. In contrast, the MEDI0457 vaccine, which contained VGX-3100 plasmid coupled with an IL-12 expression plasmid to promote T-cell function, evaluated the disease progression-free survival (PFS) at 12 months, which was estimated as 88.9% overall. These findings strengthen the hypothesis that DNA therapeutic vaccines could effectively induce de novo or boost existing immune responses. Moreover, studies have shown that using femtosecond laser treatment could also improve transfection efficiency administered intradermally and into the lesion in vivo. Thus, continuous efforts to improve the efficacy of DNA therapeutic vaccines and implementation of therapeutic vaccines into a treatment regimen as a sole approach or in combination with conservative treatment may greatly improve the current situation. Funding: There is no funding source to declare.
Institutional Review Board Statement: Due to the nature of the study (systematic review), ethical approval is not required.

Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.